Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal.
Ontology highlight
ABSTRACT:
SUBMITTER: Cho S
PROVIDER: S-EPMC1292994 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Brome mosaic virus (BMV) is a representative member of positive-strand RNA viruses. The 1a replicase from BMV is a membrane protein of unknown structure with a methyltransferase N-terminal domain and a putative helicase activity in the C-terminal domain. In order to make a functional prediction of the helicase activity of the BMV 1a C-terminal domain, we have built a model of its structure. The use of fold recognition servers hinted at two different superfamilies of helicases [superfamily 1 (SF1) and superfamily 2 (SF2)] as putative templates for the C-terminal fragment of BMV 1a. A structural model of BMV 1a in SF2 was obtained by means of a fold recognition server (3D-PSSM). On the other hand, we used the helicase motifs described in the literature to construct a model of the structure of the BMV 1a C-terminal domain as a member of the SF1. The biological functionality and statistic potentials were used to discriminate between the two models. The results illustrate that the use of sequence profiles and patterns helps modeling. Accordingly, the C-terminal domain of BMV 1a is a potential member of the SF1 of helicases, and it can be modeled with the structure of a member of the UvrD family of helicases. The helicase mechanism was corroborated by the model and this supports the hypothesis that BMV 1a should have helicase activity.
| S-EPMC7187956 | biostudies-literature
Project description:Positive-strand RNA viruses such as hepatitis C virus (HCV), flaviviruses, and coronaviruses are medically important. Assembly of replicase on host membranes is a conserved replication strategy and an attractive antiviral target. The mechanisms of replicase assembly are largely unknown, due to the technical difficulties in purifying the replicase and carrying out structural studies. Here, with an HCV replicase assembly surrogate system, we employed a bioorthogonal system to introduce the photolabile unnatural amino into each residue in the cytosolic regions of NS4B and the amphipathic helix (AH) of NS5A. Photocrosslinking enabled visualization of NS4B oligomerization and NS5A dimerization at pinpointed interacting residues and identifying contacting sites among the replicase components. Characterization of the interacting sites revealed hub elements in replicase assembly by docking replicase components to prompt protein-protein interactions. The results provide information about the molecular architecture of the replicase, advancing understanding of the mechanism of replicase assembly.
| S-EPMC8444619 | biostudies-literature
Project description:We report an RNA-negative, temperature-sensitive (ts) mutant of Murine hepatitis virus, Bristol ts31 (MHV-Brts31), that defines a new complementation group within the MHV replicase gene locus. MHV-Brts31 has near-normal levels of RNA synthesis at the permissive temperature of 33 degrees C but is unable to synthesize viral RNA when the infection is initiated and maintained at the nonpermissive temperature of 39.5 degrees C. Sequence analysis of MHV-Brts31 RNA indicated that a single G-to-A transition at codon 1307 in open reading frame 1a, which results in a replacement of methionine-475 with isoleucine in nonstructural protein 3 (nsp3), was responsible for the ts phenotype. This conclusion was confirmed using a vaccinia virus-based reverse genetics system to produce a recombinant virus, Bristol tsc31 (MHV-Brtsc31), which has the same RNA-negative ts phenotype and complementation profile as those of MHV-Brts31. The analysis of protein synthesis in virus-infected cells showed that, at the nonpermissive temperature, MHV-Brtsc31 was not able to proteolytically process either p150, the precursor polypeptide of the replicase nonstructural proteins nsp4 to nsp10, or the replicase polyprotein pp1ab to produce nsp12. The processing of replicase polyprotein pp1a in the region of nsp1 to nsp3 was not affected. Transmission electron microscopy showed that, compared to revertant virus, the number of double-membrane vesicles in MHV-Brts31-infected cells is reduced at the nonpermissive temperature. These results identify a new cistron in the MHV replicase gene locus and show that nsp3 has an essential role in the assembly of a functional MHV replication-transcription complex.
| S-EPMC2937787 | biostudies-literature
Project description:Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.
| S-EPMC4126843 | biostudies-literature
Project description:Approximately 3% of the world population is infected with hepatitis C virus (HCV), causing a serious public health burden. Like other positive-strand RNA viruses, HCV assembles replicase complexes in association with cellular membranes and produces progeny RNA genomes through negative-strand intermediates. The viral proteins required for RNA replication are nonstructural (NS) proteins NS3 to NS5B. Owing to many obstacles and limitations in structural characterization of proteins and complexes with multiple transmembrane segments, attempts to understand the assembly and action of the HCV replicase complex have been challenging. Nevertheless, great progress has been made in obtaining structural information for several replicase components, providing insights into some aspects of the viral genome replication machinery.
| S-EPMC3856683 | biostudies-literature
Project description:The carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. A serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed.
| S-EPMC230244 | biostudies-other
Project description:Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually approximately 15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.
| S-EPMC1900072 | biostudies-literature
Project description:The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3'-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2' and 3' carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn(2+) supporting about 10 times faster unwinding than Mg(2+). Unlike Mg(2+), Mn(2+) does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.
| S-EPMC2692962 | biostudies-literature
Project description:We have used an ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) to modify HCV replicase in order to identify the ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration-dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double-stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA(26). HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 and 36 min; these were absent in the control. Further we noted that both peptides were protected from FSBA modification in the presence of Mg·ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Tyr38, Tyr382, and Lys491 in catalysis, we individually substituted these residues by alanine and examined their ability to catalyze RdRp activity. We found that both Y382A and K491A mutants were significantly affected in their ability to catalyze RdRp activity while Y38A remained unaffected. We further observed that both Y382A and K491A mutants were not affected in their ability to bind template primer but were significantly affected in their ability to photo-cross-link ATP in the absence or presence of template primer.
| S-EPMC3556826 | biostudies-literature
Project description:Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing three open reading frames (ORFs). ORF1 encodes the ORF1 non-structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all three systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope ®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.
| S-EPMC8908324 | biostudies-literature